对PICK1蛋白介导的囊泡运输在癫痫失神发作中的作用机制研究

Epilapsy is one of the most common neurological disorders, from which over 9 million of patients in China suffer. Absence seizure is a general epileptic seizure which occurs more commonly in children than adults. Cummulative studies showed that metabotropic glutamate receptor mGluR7 plays an important role in absence seizure, not only from the knock out mice model but also from pharmacological studies. mGluR7 locates on the presynaptic membrane, regulates presynaptic calcium level and glutamate release. It is known that the aggragation and presynaptic membrane localization of mGluR7 is regulated by Protein interacting with C Kinase 1 (PICK1), either blocking the interaction between PICK1 and mGluR7 or knocking out PICK1 gene could cause absence-like seizures in mice, though the detailed the mechanisms requires further investigation. From our study, we have found that PICK1 interacts with Syntabulin, a protein involved in synaptic vesicle trafficking and mitochondria trafficking. We also found in heterologous cell system that Syntabulin brings PICK1 onto microtubule strucutures. The results raises the possibility that the PICK1-mediated mGluR7 transportation may also requires Syntabulin-interaction and is a microtubule-dependent trafficking event. In this study, we are going to use cultured rat hippocampal neuron as an investigation model, by using time-lapse imaging techinique, to find out the trafficking dynamic and pathway of PICK1-containing vesicles. Further combined with lentiviral mediated shRNA knock down and other biochemical techniques, we intend to examine the role of PICK1-syntabulin interaction in PICK1 localization and trafficking, and the consequent mGluR7 trafficking. The functional read out in neuronal activity would also be examined by using calcium imaging approaches. Further more, we are interested in finding out how this PICK1-Syntabulin regulated mGluR7 trafficking defect would lead to absence seizure in mice models. This study aims to unveil the underlying mechanism of PICK1-regulated mGluR7 trafficking and its functional implications in neuronal activity and absence seizure. The results would provide further clues in understanding the molecular machanisms of absence seizure,and gives a possible direction in seizure treatment.

癫痫是我国重点防治的重大疾病之一。代谢型谷氨酸受体七型（mGluR7）调节突触前钙离子浓度及神经递质释放，在癫痫失神发作中起重要作用。mGluR7聚集及膜定位受蛋白激酶Cα作用蛋白1（PICK1）调控，破坏两者相互作用或敲除PICK1基因均会引起小鼠癫痫失神发作症状，但其分子机制未被完全阐释。本课题组发现，PICK1与突触小泡运输蛋白Syntabulin作用并被转定位至细胞微管结构，提示PICK1对mGluR7的转运调节作用可能经由细胞微管上的囊泡运动完成。本课题拟在既往研究基础上，运用活细胞荧光成像技术在大鼠海马神经元内观察并分析PICK1介导的囊泡运动，探索PICK1对mGluR7的动态转运，明确Syntabulin对该过程的调节作用。本课题旨在初步提供Syntabulin和PICK1对mGluR7运输和定位的调节机制及对神经元功能影响，为进一步探讨癫痫失神发作的分子机制提供新的线索。

PICK1是一类膜周蛋白，可与多种受体相互结合并调节受体的膜表达及受体相关功能。例如在神经元内，PICK1可与兴奋性谷氨酸受体AMPA受体，代谢型谷氨酸受体mGluR7及酸敏感离子通道ASIC相互作用。此外， 越来越多研究发现PICK1可以介导始于高尔基体的囊泡运输，然而其具体机制仍不清晰。本项目研究发现PICK1与突触小泡运输蛋白Syntabulin作用并被其转定位至细胞微管结构，syntabulin可调节PICK1在以细胞微管为介质的动态转运。在培养海马神经元内，本项目发现syntabulin可促进PICK1在神经元轴突的特异表达及聚集，并介导PICK1在轴突上的动态转运。此外，研究发现syntabulin, PICK1及ASIC可形成复合物，syntabulin, PICK1及mGluR7可形成复合物，并影响ASIC介导的神经酸毒性。本项目提供了含PICK1囊泡的运输和定位的调节机制及对神经元功能影响，为进一步探讨癫痫失神发作的分子机制及神经酸毒性分子机制提供了新的线索。在此基础上，本项目发表SCI论文三篇，其中一篇发表于Journal of Neuroscience。毕业博士研究生一名，硕士研究生一名。同时该研究成果作为墙报汇报参加2015年美国神经科学年会。